A specific phosphorylation-dependent conformational switch in SARS-CoV-2 nucleocapsid protein inhibits RNA binding

The nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 encapsidates the viral genome and is essential for viral function. The central disordered domain comprises a serine-arginine–rich (SR) region that is hyperphosphorylated in infected cells. This modification regulates function, although mechanistic details remain unknown. We use nuclear magnetic resonance to follow structural changes occurring during hyperphosphorylation by serine arginine protein kinase 1, glycogen synthase kinase 3, and casein kinase 1, that abolishes interaction with RNA. When eight approximately uniformly distributed sites have been phosphorylated, the SR domain binds the same interface as single-stranded RNA, resulting in complete inhibition of RNA binding. Phosphorylation by protein kinase A does not prevent RNA binding, indicating that the pattern resulting from physiologically relevant kinases is specific for inhibition. Long-range contacts between the RNA binding, linker, and dimerization domains are abrogated, phenomena possibly related to genome packaging and unpackaging. This study provides insight into the recruitment of specific host kinases to regulate viral function.


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Secondary structural analysis of the pSR region in the non-phosphorylated form (top), PKAphosphorylated N234, SRPK1-GSK3 phosphorylated N234 (pN234(II)) and SRPK1-GSK3-CK1 phosphorylated N234 (pN234(III), bottom).Ca shifts were compared to recently proposed random coil shifts for phosphorylated peptides (66).The position of phosphorylation sites due to the different kinases are shown above in green (SRPK1), blue (GSK-3) and red (CK1).Numerous significantly shifted sites lie on a linear trajectory, suggesting that the bound form chemical shift is similar in both cases.
Bottom -13 C-1 H HMQC spectra of pN234(I) (phosphorylated using SRPK1, concentration 150 µM), free (red) and for 200% admixture of 14mer single stranded RNA (dark-blue).Six resonances appear in the region associated with 15 N-1 H cross peaks from phosphorylated residues.These peaks were assigned using standard triple resonance methods.The shading indicates regions where two out of three neighbouring residues show chemical shifts above 0.1ppm.Although small these perturbations are located on a single face of N2 (inset), suggesting that the weak interactions with N4 involve this region.

Fig. S11. Comparison of SAXS from phosphorylated and non-phosphorylated N234
SAXS of N234 in its unphosphorylated (blue), PKA phosphorylated (orange) and SRPK1/GSK-3/CK1 phosphorylated (pN234(III)) (red) states.The experimental curves are very similar, with slight differences in the pN234(III) state at inverse distances in the 1.0nm -1 range.Characteristic average radii of gyration were determined from the Guinier region of the three curves at the lowest concentration (1mg.mL - ) to be very similar (PKA phosphorylated (5.84±0.12)nm,nonphosphorylated (5.91±0.14)nmand pN234(III) (5.98±0.11)nm(fits shown below).Intensity build-ups for directly phosphorylated amino acids (blue) and neighbouring or nearneighbouring residues (red) are similar.Similarly, the increase in intensity of the sidechain methyl group of T198 mirrors the behaviour of the backbone 15 N-1 H correlation peak.These comparisons provide support for the assignment and the kinetics of phosphorylation derived from the analysis of the 15 N-1 H correlation peaks of the directly phosphorylated residues.
Fig. S3.Phosphorylation by SRPK1 of N234 does not impact RNA-binding.

Fig. S7 .
Fig. S7.Comparison of RNA-binding to N123 and N234.Chemical shift perturbations (CSP) measured in the 15 N-1 H TROSY spectrum of N234 following addition of single stranded 14mer RNA (blue) in comparison with CSPs measured in the 15 N-1 H TROSY spectrum of N123 following addition of the same single stranded 14mer RNA (semitransparent red).In the common regions (N2, N3), only residues for which CSPs were measured in both mixtures are shown.This comparison indicates that N1 and N4 do not strongly modify the RNA binding mode on N2.

Fig. S9 .
Fig. S9.Comparison of intensities of phosphorylated and non-phosphorylated peaks.A -Comparison of 15 N-1 H HSQC of peak intensities for resonances corresponding to the SR region of N234 prior to (blue) and post (red) phosphorylation by SRPK1 and GSK-3 (pN234(II)), indicating that phosphorylation is essentially complete (within the signal to noise of the experiment).B -Comparison of increase in intensity of the 15 N-1 H SOFAST HSQC of the peak corresponding to pS184/pS202 and the decrease in intensity of S202.Both curves were simultaneously fit to a single exponential describing the increase and decrease of the intensities respectively.
Fig. S10.Comparison of 15 N-1 H HSQC chemical shifts of N2 in its isolated form and in constructs containing N1 and N3 and N4.Red -difference in chemical shifts of N2 and N2 in N234 Green -difference in chemical shifts of N2 in N234 and N2 in N123

Fig S12 .
Fig S12.Impact of phosphorylation on N234-RNA assembly (A) Negative staining electron microscopy of N234 in complex with 14mer RNA showing cagelike particles associated with encapsidation substructures.(B) Such particles are absent in mixtures of RNA with phosphorylated N234.